Project Summary/Abstract Obesity-associated insulin resistance is a hallmark of type 2 diabetes (T2D) and plays a central role in metabolic syndrome. Numerous studies suggest a firm connection between obesity-induced inflammation and insulin resistance mediated by macrophages and other immune cells. A recent elegant study by Olefsky and his team [Cell publication PMID: 27814523 ] shows that galectin-3 (Gal3), a beta-galactoside-binding lectin, is a macrophage-derived instigator of ?insulin resistance and impaired glucose tolerance?, associated with obesity- induced T2D. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. The study shows that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These fundamental observations elucidate a novel role of Gal3 that promotes obesity-mediated inflammation (macrophage-derived Gal3) and insulin resistance and suggest that specific inhibition of Gal3 may represent a promising therapeutic strategy to restore insulin sensitivity. Our scientific premise is that we have developed a very potent Gal3 antagonist, named TFD100, from a natural dietary source [PNAS publication, PMID 23479624]. TFD100 specifically targets Gal3 with picomolar affinity ? the affinity is so high (10-100-fold more) to counteract Gal3?s natural affinity to its intrinsic ligands such as IR. In our preliminary studies, Gal3 inhibited IR activation of the engineered CHO/IR/IRS-1 cells, which was reversed by TFD100. Our research team has extensive expertise in a relevant mouse model of T2D ? a high fat diet (HFD)-induced model in C57BL/6 (B6) . We demonstrated increased expression of Gal3 in liver of HFD fed mice compared to that of low fat diet (LFD) fed mice. Gal3 is also high in obese mice. Moreover, Gal3 levels in sera of T2D patients are found significantly linked with indices of insulin resistance. Based on the preliminary data we hypothesize that that specific inhibition of Gal3 with TFD100 will interfere with Gal3-IR interactions on myocytes, adipocytes and hepatocytes and thereby restore insulin sensitivity. We will test this hypothesis in the following specific aim: Investigate the therapeutic utility of TFD100 for treating T2D in a relevant mouse model. First we will determine TFD100?s ability to impede Gal3-mediated cellular insulin resistance. Various cells such as adipocytes, myocytes, hepatocytes, and engineered CHO/IR/IRS-1 cells will be treated with TFD100 and determine its effect on IR activation, signaling, and glucose uptake. Next, we will determine TFD100?s ability to treat HFD-induced obesity, insulin resistance, and T2D in both male and female mice. After drug (TFD100) treatment, glucose and insulin tolerance (primary endpoint) will be measured. For other endpoints, resolution of inflammation and restoration of insulin signaling will be measured by the frequency of pro-inflammatory biomarkers (gene and protein) in blood, liver, muscle and fat. This also includes determination of changes in pro-inflammatory immune cell frequencies such as polarization of macrophages. This study, for the first time, will explore the therapeutic utility of a very potent natural compound that outcompetes Gal3?s intrinsic interaction with the IR to reverse insulin resistance.